In early preclinical drug development, it is necessary to carry out comparative analyses of the effect of substances on cells in vitro. For this purpose, cell cultures need to be prepared under strictly controlled conditions and treated with the therapeutic agent of interest. Reliable results can only be obtained, if individual (i.e. identical or different) cell cultures are tested in a reproducible and highly comparable manner.
But, often serious problems are encountered. For example, cell cultures are damaged or put under stress during the cell culture preparation or testing procedures, and thus, the analysis may show results that are at least in part a consequence of such damage. Moreover, applying conclusions drawn from results obtained on damaged cells to the situation in vivo may cause fatal errors. Furthermore, the damages or stressful conditions are not reproducible between individual cell cultures and may influence a variety of factors, potentially leading to a high percentage of false positives or false negatives.
For this reason, in vivo tests in animal model systems subsequent to in vitro tests are very important. This leads to the paradox that the validity expected for experiments in human cell culture themselves cannot be realized due to uncontrolled testing conditions. The expected validity is foiled by experimental artefacts.
On the other hand, in vivo results obtained by animal studies are less afflicted by experimental artefacts. However, they have per se only limited validity for human applications (not to mention the problem of breeding genetically identical animal cohorts for comparative analysis).
For this reason, efforts have been made, e.g., by starting highly expensive genome and proteome projects, to assure the transferability of results from animals to humans. However, a satisfying solution to this problem is still far from being achieved.
Therefore, at present the risk of new pharmaceutical developments still largely pertains to the field of clinical studies, which is the most expensive and ethically most problematic field. Similar problems also exist in the development of diagnostic tests on cell cultures.
The invention thus seeks to develop a culture device and methods for testing in vitro that minimize stressful conditions on the living cell material. This will permit to increase the validity of results obtained by comparative analyses, and preferably, these may be directly extrapolated to the whole organism that corresponds to the cultured cells. Thereby, it may be possible to avoid or reduce the number of further animal tests.
All the components of cosmetics, pharmaceutical drugs and chemical consumer products require testing for a broad spectrum of side effects. The ultimate goal of the risk assessment strategy is to define the use and application of the product to minimize health and environmental hazards. In addition to, e.g., toxic, corrosive, carcinogenic effects and embryo toxicity, immunogenicity has to be taken into account for product safety. Product-related immunogenicity may cause, e.g., skin sensitization, allergy and anaphylaxis.
Immunogenicity is only desirable for the purpose of vaccination. Pharmaceutical drugs, cosmetic products and other consumer-related chemicals including food ingredients, as well as combinations thereof may cause unexpected immunogenicity when applied to the human body. Thus, immunogenicity and altered immunofunction is a fundamental problem associated with the use of chemicals and biopharmaceuticals.
Product-related parameters such as drug design, manufacturing process, formulation or route of administration may have an influence on immunogenicity (Schellekens, H., Bioequivalence and the Immunogenicity of Biopharmaceuticals. Nature Reviews Immunology. Vol. 1, 457; 2002).
New chemical entities, but also biopharmaceutical drugs such as recombinant proteins, animal- and plant-derived components may cause the generation of neutralizing antibodies, allergic reactions and anaphylaxis in the patient. New chemical entities, but also biological substances, such as antibodies or cytokines react and interfere highly specifically with a certain target, or have certain species specificity in their mode of action.
Therefore, for testing immunogenicity and immunofunction, model systems need to be developed that closely mimic the situation in humans. Thus, in order to study these effects, equivalent test systems and robust procedures based on human immune competent lymphatic organoids are needed.
A number of in vitro tests using human cells are available, but they do not emulate organ- or tissue-functionality, and thus, are of limited value. Therefore, for the investigation of drug efficacy and adverse effects, in vivo tests using animal species have been absolutely necessary.
A large panel of validated animal testing systems are established and mandatory for product approval, especially in the field of pharmaceuticals, chemical and cosmetic industry.
For early pre-clinical studies as well as late drug screening procedures, a number of transgenic animal models have been developed and are already available for testing of induction of immune responses. Well-established animal models are mandatory for late pre-clinical toxicity testing (e.g. mice, rat, dog and non-human primates). Within the scope of a clinical trial, immunogenicity testing in humans is focussing on the analysis of blood and urine samples of treated volunteers for drug-neutralising antibodies.
Humanized animal models have been described in, e.g., WO 2006/056769 A1, providing mice transgenic for human MHC class II. WO 02/102830 A1 describes animal models, which supplement or replace the natural albumin sequence with a typical human serum albumin sequence. U.S. Pat. No. 6,248,721 provides humanized animal models for the evaluation of vaccines designed to confer immunity against human pathogens, including vaccines directed against the human immunodeficiency virus.
Other approaches have aimed at the identification of T cell epitopes. US 2004/0180386 A1 describes a method for epitope mapping (screening) using peptide libraries of overlapping sequences to design new proteins with reduced immunogenicity.
Moreover, a number of data libraries based on computational models have been generated in order to estimate the probability of antigen matching to known human relevant epitopes. For example, U.S. Pat. No. 6,939,546 B2 describes a computer-based model for binding studies of peptides to Class II MHC-receptors. Some predictive information about the immunogenic potential of peptides can be generated. These data can help to reduce the number of in vitro tests that need to be done subsequently.
Methods have been described for testing immune functions with the help of isolated animal and human cells. Mostly isolated peripheral blood mononuclear cells (PBMC) of different species are cultured in suspension and exposed to drugs in different concentrations. Induced cell proliferation and cytokine release is monitored over a short period of, e.g., 1 to 48 hours.
For a more detailed investigation, defined subpopulations of PBMC are used for the analysis of cell-type specific responses. T cells, for example, are used for peptide and epitope mapping and dendritic cells are used to analyse the presentation of antigens. US 2003/0152550 A1 describes the use of dendritic cells in screening and testing of drugs affecting dendritic cell maturation.
Common read-out parameters are antigen-dependent proliferation of primed lymphocytes and antigen-dependent cytokine secretion.
A major drawback of existing in vitro tests is that they are carried out on a suspension of cells in a test tube, whereas most of the physiological reaction in the body is tissue-related and organ-related. Secondary lymphatic organs and all solid body tissues like skin are the structural and environmental basis for most of the immune reactions and not the peripheral blood. Therefore, artificial tissue models which emulate tissue or organ functionality are needed.
Bioartificial organs have been developed for the purpose of fluid processing (US 20050142530). Other systems have aimed at providing tissue-engineered systems (comprising liver tissue, kidney tissue, cardiac tissue, cartilage tissue, or bone marrow tissue) for testing drug metabolism and toxicity (WO 2004065616 A2, WO2003104439 A2). US20060110822 describes a multiwell-based perfusion flow bioreactor for drug testing on cells in dynamic cell cultures.
For suitable tissue culture techniques, it has become obvious that, in addition to efficient oxygen and nutrient supply, the establishment of local gradients of (i) metabolites, (ii) cytokines, and (iii) chemokines and other (undiscovered) parameters, as well as structured surfaces for chemotaxis and local settlement (including intercellular cross-talk via tight junctions), are crucial prerequisites for the proper emulation of in vivo environments (Griffith, L. G. and Swartz, M. A. 2006. Capturing complex 3D tissue physiology in vitro. Nat Reviews Molecular Cell Biology, 7: 211-224). This provoked a shift from the development of homogeneous culture systems to heterogeneous ones and an emphasis on controlled, continuously adjustable, long-term culture processes.
The basic aims of those cell culture devices and process developments are to create an architecture and homeostasis mimicking the specific relevant human microenvironment for self-organisation of a specific tissue (see US 2005/0142530 A1).
Human tissue based models that emulate immune organ function are conceived to bridge the gap between early lead optimization and the pre-clinical development stage. Human or animal lymphatic organoid models may provide insights into the mode of drug action and, in addition, can be used to refine a product related risk profile.
A technological platform for the emulation of human immune function in vitro using human cells in a tissue-like (organoid) arrangement and robust testing procedures give the opportunity for predictive in vitro testing of immunogenicity and human immune functionality. It can be used for optimised product development and better patient and consumer health and safety. In addition, the technology and procedures give the chance for reduction and replacement of animal testing.
Attempts have been made to simulate the vaccination process in vitro in order to investigate vaccine candidates for their mode of action and their potency. For this purpose, a modular miniaturized immunobioreactor system (WO 2005/104755 A2) has been developed, which comprises a lymphoid tissue equivalent. The lymphoid tissue equivalent is created by seeding T and B cells onto microcarriers, and cocultivating T and B cell populated microcarriers in a porous container.
In the prior art, most of methods for tissue engineering are based on adherent cells. Cell culture methods for non-adherent cells normally use suspension cultures. A disadvantage of suspension culture is that cells in suspension culture are single cells that do not emulate tissue or organ functionality. Suspension cultures further suffer from the problem that it is difficult to withdraw samples without removing suspended cells from the culture, without using tedious procedures such as centrifugation and without interrupting the incubation. Therefore a cell culture system for non-adherent cells having the advantages of cell culture systems for adherent cells would be very desirable.
In general, interruption of the incubation of cells exerts stress on the cells, which may bear the risk of measuring artefacts due to such stress. Therefore, for comparative analysis, a culture method allowing to analyse the effect of a test compound on cells without interrupting the incubation of cells is needed.